Growth medium for human corneal endothelial cells

ABSTRACT

A human cell growth medium formulation for culturing human cells of neural crest origin, most preferably corneal endothelial cells, or for accelerating the growth and proliferation of human cells of neural crest origin is disclosed. The formulation for the nutrient medium of the invention includes nerve growth factor, preferably at a concentration of 1-100 ng/ml and most preferably 20 ng/ml. The growth medium formulation preferably also includes epidermal growth factor (preferably at a concentration of 1-200 ng/ml and most preferably 5 ng/ml). Most preferably, the formulation of the invention also includes fibroblast growth factor (pituitary), at a concentration of 5-400 ng/ml or, most preferably, 40 ng/ml,

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority of U.S. Provisional PatentApplication No. 60/119,788 filed, Feb. 11, 1999 entitled GROWTH MEDIUMFOR HUMAN CORNEAL ENDOTHELIAL CELLS, the whole of which is herebyincorporated by reference herein.

STATEMENT REGARDING FEDERAL FUNDING

Part of the work leading to this invention was carried out with UnitedStates Government support provided under Grant No. NEI R01 EY05767 fromthe National Eye Institute. Therefore, the U.S. Government has certainrights in this invention.

BACKGROUND OF THE INVENTION

Corneal endothelial cells are different from vascular and pulmonary“endothelial cells” as they have a different embryonic tissue origin.Human corneal endothelial cells do not normally proliferate in vivo toreplace those lost due to cell injury or death. Growth of these cells inculture is also extremely limited. This can be a serious problem as age,diseases such as glaucoma and diabetes, and ocular surgical procedures,such as laser vision correction and cataract extraction and intraocularlens implantation, cause an accelerated loss of these precious cells.There are no medical treatments for corneal diseases resulting fromendothelial cell loss. Currently, corneal transplantation is the onlyway to restore normal vision.

The relative ability of corneal endothelial cells to proliferate in vivoand in culture appears to be a function of age; i.e., embryonic cornealendothelial cells and cells from neonates will proliferate much morereadily than cells from children and adults. In a few cases, researchershave been able to culture cells from older donors, but growth has beensupported by seeding the cells onto an artificial matrix, such aschondroitin sulfate/laminin, or onto extracellular matrix secreted bycorneal endothelial cells from cows, one of a number of species whosecorneal endothelial cells do grow readily in culture. A reliable way ofsupporting cell culture of human corneal endothelial cells would behighly desirable.

BRIEF SUMMARY OF THE INVENTION

A new formulation for a culture medium that can be used to overcome suchdifficulties has now been developed. In general, the invention isdirected to a formulation for a growth medium for culturing cells ofneural crest origin, most preferably corneal endothelial cells, or foraccelerating the growth and proliferation of such cells. The formulationfor the nutrient medium of the invention includes nerve growth factor,preferably at a concentration of 1-100 ng/ml and most preferably 20ng/ml. The growth medium formulation preferably also includes epidermalgrowth factor (preferably at a concentration of 1-200 ng/ml and mostpreferably 5 ng/ml). Most preferably, the formulation of the inventionalso includes fibroblast growth factor, at a concentration of 5-400ng/ml or, most preferably, 40 ng/ml.

A commercial growth medium, particularly a serum-free medium, forms auseful basis for the growth medium of the invention. The growth mediumof the invention can be augmented with serum, e.g., fetal bovine serum,as needed. As is well known to cell culture specialists, a useful humancell growth medium preferably also includes added ascorbic acid, humanlipids, antibiotics and cell viability stabilizers.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims, taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a cross-section of a cornea showing the orientation of thecorneal layers;

FIGS. 2A and 2B are micrographs showing comparative examples of thegrowth of human corneal endothelial cells from older donors using aprior art formulation (FIG. 2A) and the growth medium of the invention(FIG. 2B);

FIG. 3 is a bar graph showing cell density of transplanted corneas. Aline drawn at 500 cells/mm² indicates the cell density below which thereare insufficient cells to maintain corneal clarity;

FIG. 4 is a graph showing pachymetry of transplanted corneas afterouabain treatment following 14 days in organ culture to illustrate thattransplanted endothelium is functional; and

FIGS. 5A and 5B are micrographs comparing the morphology of normal andtransplanted human corneal endothelium.

DETAILED DESCRIPTION OF THE INVENTION

The cornea is the “window” of the eye. Because it transmits light intothe eye and protects the eye from the environment, normal visionrequires a clear cornea. Corneal clarity results from the specificarrangements of fibrils in the stromal layer (see below) that permitlight to be transmitted through the cornea rather than to be absorbed.If water is present in the stroma in excess amounts (corneal edema), thearrangement of these fibrils is disrupted, resulting in corneal cloudingand loss of visual acuity.

As shown in FIG. 1, the cornea consists of four major layers. Theepithelium is the outermost layer of the cornea. It is 5-7 cells thickand protects the eye in a manner similar to that of skin. It is bathedon the outside by tears. The stroma is the thick, gel-like middle layerthat contains numerous long filaments separated from each other bymolecules which tend to attract water. These filaments are preciselyarranged to let light pass through the cornea without being bent orabsorbed. The stroma also maintains the cornea in the curved shaperequired to focus light on the lens and the retina.

Bordering the interior of the stroma is a thick acellular layer known asDescemet's membrane. Endothelial cells secrete the gel-like material ofthis layer, and the composition of this layer differs from that of thestroma. Descemet's membrane is the substrate to which the endothelialcell layer is attached. The endothelium forms the innnermost layer ofthe cornea and is just one cell thick. The base of this cell layer isattached to Descemet's membrane and the surface is bathed by aqueoushumor, the nutrient fluid that occupies the anterior chamber of the eye.

The cornea does not contain blood vessels and, as is such, nutrition ofthe corneal tissues occurs through both the ocular surface (whichsupplies the majority of oxygen) and the aqueous humor (which suppliesall other nutrients). Cells of the endothelial monolayer permit slowpercolation of aqueous humor through the intercellular spaces into thestroma, but the arrangement of these cells is sufficiently tight to forma barrier to prevent excess fluid from entering the stroma. Endothelialcells also contain an “ionic pump” in their plasma membrane thatactively removes excess water from the stroma and returns it to theaqueous humor.

In spite of their importance in maintaining normal vision, cornealendothelial cells do not divide to replace dead or injured cells. So,throughout life, human endothelial cells are lost but not replaced.Instead, the remaining cells enlarge to cover the area formerly occupiedby the dead or injured cells. As humans age, this gradual cell loss andenlargement of the remaining cells can cause breakdown of theendothelial barrier. In addition, diseases such as glaucoma anddiabetes, and ocular surgical procedures, such as laser visioncorrection and cataract extraction and intraocular lens implantation,cause an accelerated loss of these precious cells. When cell density isreduced below a critical number (e.g., 500 cells/mm²), water enters thestroma and disrupts the filament arrangement. This disruption causes thecornea to become cloudy. The visual loss resulting from corneal cloudingis a form of blindness. Light can be perceived, but images are blurred,giving the appearance of looking through a dense fog. There are nomedical treatments for corneal diseases resulting from endothelial cellloss. Currently, corneal transplantation is the only way to restorenormal vision. The growth medium of the invention makes possibleimportant new approachs to correcting these problems.

The optimal formulation of the growth medium of the invention wasdetermined by dose-dependent testing of each ingredient on the viabilityand/or ³H-thymidine uptake (a measure of DNA synthesis/proliferation) ofcultured rat corneal endothelial cells (a species whose cells grow wellin culture). As the formulation developed, it was used to culturecorneal endothelial cells from adult (>50 y.o.) human donors. For thisculture, Descemet's membrane and attached endothelial cells weremechanically stripped from the donor cornea. The resulting tissue pieceswere incubated for 1 hour in 0.2 mg/ml ethylenediaminetetraacetic acid(EDTA) to remove endothelial cells from Descemet's membrane. Aftercentrifugation, the isolated cells were placed in 6-well tissue cultureplates and incubated in culture medium at 37° C. in a 5% CO₂ humidifiedatmosphere. Medium was changed every other day. Cultured cells reachedconfluence in 7 to 14 days.

Prior to the development of this formulation, studies were conducted totest the ability of adult corneal endothelial cells to grow on anartificial matrix (1% chondroitin sulfate/1% collagen) and on bovine orrabbit corneal endothelial cell matrix. Better growth was obtained onthe artificial matrix, but cell shape frequently differed from normal.Once the preferred formulation of the growth medium was determined, itwas possible to grow cells from older adult (>50 y.o.) donors isdirectly on tissue culture plastic and still have them retain theirnormal polygonal shape. Thus, the use of the growth medium of theinvention does not require coating the plastic with either artificialmatrix or cell-derived matrix as is required by prior art formulations.FIG. 2 shows the results when endothelial cells from older cornealdonors were cultured for the same period of time in either Medium 199(GIBCO/BRL) supplemented with 10% fetal calf serum and 1% chondroitinsulfate and plated on a 1% collagen matrix (A) or were cultured with thegrowth medium of the invention on tissue culture plastic (B). Cells in(A) exhibit a lower density and abnormal, elongated morphology. Cellsgrown in the growth medium of the invention grow to a higher density andexhibit normal, polygonal morphology.

The preferred formulation of the growth medium of the invention has thefollowing composition:

1. OptiMEM (containing insulin, transferrin, and selenium)—OptiMEM is acommercially available serum-free medium.

2. 8% fetal bovine serum (Hyclone) preferred range 125%.

3. 20 μg/ml ascorbic acid (Sigma)

4. 0.005% human lipids (Sigma)

5. 40 ng/ml fibroblast growth factor (pituitary) (Biomedical ResearchTechnologies)—preferred range from 5-400 ng/ml

6. 5 ng/ml epidermal growth factor (mouse)—(UpstateBiotechnologies)—preferred range from 1-200 ng/ml

7. 20 ng/ml Nerve Growth Factor—(Biomedical Research Technologies)preferred range from 1-100 ng/ml

8. 0.08% chondroitin sulfate (Sigma)

9. 200 μg/ml calcium chloride (Sigma)

10. 50 μg/ml gentamycin (GibcoBRL)

11. RPMI-1640 Multiple Vitamin Solution- {fraction (1/100)} (Sigma)

12. Antibiotic Antimycotic Solution- {fraction (1/100)} (Sigma)

The growth medium of the invention differs from prior formulations in anumber of specific ways. For example, the base medium used in thepreferred formulation, OptiMEM (GIBCO/Life Technologies), containsingredients important for supporting cell proliferation, such asinsulin, transferrin, and selenium. Other nutrient media containingthese three ingredients could substitute for OptiMEM. The growth mediumof the invention also contains nerve growth factor, which is generallynot included in culture media. Preferably, the medium of the inventioncontains a combination of nerve growth factor (NGF), epidermal growthfactor (EGF) and fibroblast growth factor (FGF). Additionally, thepreferred FGF concentration, at 10 ng/ml, is significantly lower thanthe 400 ng/ml FGF concentration in other conventional media, e.g.,Englemann's medium.

The following example is presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. It is not intended in any way otherwise to limit thescope of the disclosure.

EXAMPLE 1

Evaluation of the morphology and function of cultured human cornealendothelial cells transplanted onto denuded Descemet's membrane of donorcorneas.

Corneas from donors (52-80 years old) were obtained from an eye bank.Descemet's membrane was stripped and endothelial cells were removed bymild EDTA treatment. Cells, cultured in the preferred formulation of theculture medium, were identified by RT-PCR of collagen type VIII andkeratin-12 and by morphology. Cells (2.5×10⁵) were seeded onto denudedDescemet's membrane from a second donor cornea. The cornea was incubatedin culture for up to 1 month. Cell-cell contact of transplanted cellswas evaluated by ZO-1 staining. Morphology and ultrastructure wereexamined by transmission electron microscopy. Pump function was testedby pachymetry after exposure to 200 μM ouabain. Cultured cells wereidentified as corneal endothelium by characteristic mRNA expression andmorphology. The success rate of primary cultures, obtained from corneasstored <5 days after death, was about 99%. Cultures became confluentwithin one week. ZO-1 staining revealed normal cell shape and cell-cellcontacts compared to normal corneas. Furthermore, as can be seen in FIG.3, after 14 days in organ culture, the cell density (number ofcells/mm²) of the transplanted corneas is within the range of both thenormal donor and normal recipient corneal cell densities.

With time, cell-substrate adhesion, cell-cell contact, and lateralinterdigitation increased, and the cells rearranged to form a monolayer.When the endothelium is functional, it draws water out of the cornealstroma, resulting in a thin cornea. Ouabain, a cardiac glycoside,inhibits the function of the endothelial “ionic pump”, causing anincrease in corneal thickness. Upon exposure of the transplantedendothelium to ouabain, corneal thickness increased to a level equal tothat of corneas denuded of endothelium, as measured by pachymetry.Referring to FIG. 4, pachymetry of transplanted corneas after ouabaintreatment is graphically depicted. The open triangles representthickness measurements from a cornea that was denuded of endothelialcells (negative control). Without a working “pump”, this cornea remainsthick (about 950 μm). The other symbols represent data from 5 corneascontaining transplanted endothelial cells (transplants were maintainedfor 14 days in organ culture prior to thickness measurements). Withexposure to ouabain, corneal thickness increased over a 2-3 hour periodand reached the maximum thickness exhibited by the negative control. Theability of ouabain to increase corneal thickness indicates that theendothelium in the transplants was functional prior to ouabain addition.

The various determinations described here show that the morphology andfunction of transplanted human corneal endothelial cells from olderindividuals are similar to those of normal endothelial cells. Referringto FIG. 5, thick plastic sections are shown comparing the morphology ofnormal and transplanted human corneal endothelium. The micorgraph on theleft is a section of normal cornea obtained from a 61 year old donor.The micrograph on the right shows a cornea from a 63 year old donor thathas been cultured in the growth medium of the invention. Thetransplanted cornea was photographed after 14 days in organ culture.After this time in culture, a normal monolayer of cells can be seenassociated with the recipient Descemet's membrane. The density of thetransplanted cells is similar to that of normal endothelium. (Originalmagnifications: Left micrograph=40×, right micrograph=63×.)

Use

The growth medium of the invention may be applied to a patient's eye astopical drops or injected into the anterior chamber to induce transientproliferation of corneal endothelial cells in older individuals whosevisual acuity is impaired due to low endothelial cell counts.Furthermore, cells from an older individual having low cornealendothelial cell density could be cultured in the growth medium of theinvention to increase cell numbers. These could then be re-seeded ontothe cornea of the patient to increase cell density and improve visualacuity.

The growth medium of the invention may be used as an irrigating solution(most probably not including serum) during anterior chamber surgicalprocedures to maintain the overall health and stability of the cornealendothelium (which can be compromised during these procedures) and topromote proliferation to replace cells damaged during the procedure.Furthermore, the growth medium of the invention (also without serum)would be a better supportive medium for the storage of donor corneasprior to transplantation than the prior art media.

The growth medium of the invention can support growth of human cornealendothelial cells for use in the development of “artificial” corneas. Upto this time, experimental artificial corneas have been prepared eitherwith endothelial cells from species that grow better, such as mouse, orwith SV40 transformed corneal endothelial cells.

With the use of the medium of the invention, researchers can now growadult human corneal endothelial cells in culture without the need toseed cells onto artificial or cell-derived matrices. This will provide aready supply of cells for molecular and cell biological studies thathave been impossible to perform up to now.

Other Embodiments

The tissue embryonic origin of the corneal endothelium is neural crest.During fetal development, neural crest cells migrate and thendifferentiate to form many types of tissue. Many of the cell typesformed from neural crest do not readily proliferate in vivo. The growthmedium of the invention would be useful to induce proliferation in otherocular cells of similar origin, including, but not limited to, lacrimalgland acinar cells, ciliary body epithelium, corneal stromalkeratocytes, skeletal muscle of the dorsal iris, and trabecular meshworkepithelium. Other non-ocular cells of similar embryonic origin shouldalso proliferate upon exposure to this medium. Among non-ocular celltypes of neural crest origin are sensory neurons, Schwann cellsassociated with the peripheral nervous system, and smooth muscle cellsassociated with the branchial arch arteries. Thus, the growth medium ofthe invention could be useful for repair of damaged tissue made up ofthe indicated cells, such as peripheral nerve damage and peripheralskeletal muscle damage, or for treating heart muscle after infarction.

The preferred formulation of the growth medium of the invention can bevaried by those of skill in the art without compromising the positiveeffects of the invention as long as the formulation used includes nervegrowth factor (NGF).

While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

What is claimed is:
 1. A human cell growth medium formulation forculturing human cells of neural crest origin or for accelerating thegrowth and proliferation of human cells of neural crest origin, saidgrowth medium formulation comprising: insulin; transferrin; selenium;1-100 ng/ml nerve growth factor; 5-400 ng/ml fibroblast growth factor(pituitary); 1-200 ng/ml epidermal growth factor; 1-25% fetal bovineserum; 10-50 μg/ml ascorbic acid; 0.001-0.01% human lipids; 0.01-0.12%chondroitin sulfate; 100-300 μg/ml calcium chloride; 10-100 μg/mlgentamycin; RPMI-1640 multiple vitamin solution ({fraction(1/50)}-{fraction (1/200)}); and antibiotic antimycotic solution({fraction (1/50)}-{fraction (1/200)}).
 2. The growth medium formulationof claim 1, said growth medium formulation comprising about thefollowing concentrations: 20 ng/ml nerve growth factor; 40 ng/mlfibroblast growth factor (pituitary); 5 ng/ml epidermal growth factor;8% fetal bovine serum; 20 μg/ml ascorbic acid; 0.005% human lipids;0.08% chondroitin sulfate; 200 μg/ml calcium chloride; 50 μg/mlgentamycin; RPMI-1640 multiple vitamin solution ({fraction (1/100)});and antibiotic antimycotic solution ({fraction (1/100)}).